Structural and electronic characterization of heme moiety in oxygenated hemoproteins by using XANES spectroscopy

Abstract

Iron K-edge X-ray absorption near edge structure (XANES) spectra were measured for oxy-forms of cytochrome P-450 cam ( P-450 cam), horseradish peroxidase (HRP) and myoglobin (Mb) by using Synchrotoron Radiation of Photon Factory (Tsukuba). A pronounced 1s-4p transition and some fine structures were well-resolved in the spectra obtained. Comparing the spectra, the features at the fine structures termed P, C and D, were similar among the three hemoproteins, suggesting a similar site-symmetry around the heme iron and the same FeOO bond angle (about 115°). On the other hand, absorption features at the edge region (7115–7135 eV) were slightly but significantly different from one another; the absorption intensity at 7115–7135 eV region increased in the order of Mb, HRP and P-450 cam, while that at 7125–7135 eV decreased in the same order. A similar absorption feature was also obtained with their deoxy (ferrous high spin) forms. We assumed that the absorption at the lower energy region (7115–7125 eV) reflects the π-character in the Fe-ligand bond, whereas that at the higher energy region (7125–7135 eV) does the δ-character, on the basis of the previous and comprehensive studies of the XANES spectroscopy of the adsorbed molecules on the metal surface (McGovern et al. (1989) Handbook on Synchrotoron Radiation, Vol. 2, pp. 467–539). According to our assumption, our XANES results indicated that the π-character of the Fe-ligand bond increases in the order of Mb, HRP and P-450 cam, and that the π-electron of the thiolate S − in P-450 cam is donated to the FeOO moiety, most probably to the antibonding π ∗ orbital of O 2. Such an interpretation is consistent with the experimental findings or data accumulated so far by other methods, such as the resonance Raman spectroscopy.